D-enzyme compositions and methods of their use

ABSTRACT

D-enzyme compositions are described comprising an amino acid residue sequence that defines an polypeptide able to catalyze an enzymatic reaction. The D-enzyme has an amino acid residue sequence consisting essentially of D-amino acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/343,585, filed Dec. 2, 1994 now U.S. Pat. No. 6,548,279, which is aNational Phase of International Application No. PCT/US93/05441, filedJun. 7, 1993 which is a continuation-in-part of U.S. patent applicationSer. No. 07/894,817, filed Jun. 8, 1992 now abandoned. The disclosuresof all the above patent applications are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to proteins incorporating D-amino acidresidues. More particularly, the present invention relates toenzymically active proteins consisting essential of D-amino acids andmethods for using such proteins.

BACKGROUND

The biosphere is inherently chiral; each class of biologicalmacromolecules is rade up of monomer molecules of uniform chirality(Mason, Chirality 3:223, 1991) and the biochemical interactions ofbiological macromolecules are inherently chiral.

Enzymes, for example, invariably act only on one enantiomer of a chiralsubstrate, or generate only one diastereomer from a prochiral substrate.Fersht, in “Enzyme Structure and Mechanism”, W.H. Freeman and Company,San Francisco, 1977, pp. 75–81. This specificity can be related to thechiral structure of the enzyme molecule, including the three-dimensionalfolding of the polypeptide backbone and the orientation of the aminoacid side chains in the folded protein molecule. Fersht, supra. To dateonly L-enzymes have been described in nature; this leaves thedescription of D-enzymes and their properties, which include foldedstructure, enzymatic activity, and chiral specificity, as unexploredquestions.

Recently, Zawadzke et al., J. Am. Chem. Soc., 114:4002–4003, 1992,described the preparation of a small 45 amino acid residue polypeptide(D-rubrodoxin) using D-amino acids. L-rubrodoxin is found in clostridiaand is the simplest iron-sulfur protein. It is believed to function inelectron transport. However, it lacks an demonstrated enzymic activity.

Prior to the present invention, the largest L-protein known to bechemically synthesized in a conventional step-wise fashion isPreprogonadotropin Release Hormone (PreproGnRH). PreproGnRH has 93 aminoacid residues. (Milton et al., Biochemistry, (1992) 31: 8800.)PreproGnRH inhibits prolactin release.

Many organic compounds exist in optically active forms, i.e., they havethe ability to rotate the plane of plane-polarized light. In describingan optically active compound, the prefixes D and L or R and S are usedto denote the absolute configuration of the molecule about its chiralcenter(s). The prefixes (+) and (−) or d and l are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they aremirror images of one another. A specific stereoisomer may also bereferred to as an enantiomer, and a mixture of such isomers is oftencalled an enantiomeric or racemic mixture.

The property of optical activity is due to molecular asymmetry aboutcarbon atoms that are linked to four different atoms or molecules. Wherethere is only one asymmetric carbon atom, or chiral center as it issometimes called, there are two possible stereoisomers. Where there aren asymmetric carbons or chiral centers, the number of potentialstereoisomers increases to 2^(n). Thus, a molecule with three chiralcenters would have eight possible stereoisomers.

While the structural differences between stereoisomers are subtle and oflittle consequence in ordinary chemical reactions, they may be profoundwhere biological systems are concerned, i.e., if the compounds areutilized in enzyme-catalyzed reactions. Thus, the L-amino acids arereadily metabolized in humans but the corresponding D-analogs are not,and only D-glucose can be phosphorylated and processed into glycogen ordegraded by the glycolytic and oxidative pathways of intermediarymetabolism. Similarly, beta blockers, pheromones, prostaglandins,steroids, flavoring and fragrance agents, pharmaceuticals, pesticides,herbicides, and many other compounds exhibit critical stereospecificity.In the field of pesticides, Tessier [Chemistry and Industry, Mar. 19,1984, p. 199] has shown that only two of the eight stereoisomers ofdeltamethrin, a pyrethroid insecticide, have any biological activity.The same statement concerning the concentration of bioactivity in asingle isomer can be made about many other pesticides, including thephenoxypropionates and halopropionate derivatives, each containing onechiral center and existing in the form of two optical isomers.

Stereochemical purity is of equal importance in the field ofpharmaceuticals, where 12 of the 20 most prescribed drugs exhibitchirality. A case in point is provided by naproxen, or(+)-S-2-(6-methoxy-2-naphthyl)-propionic acid, which is one of the twomost important members of a class of 2-aryl-propionic acids withnon-steroidal anti-inflammatory activity used, for instance, in themanagement of arthritis. In this case, the S(+) enantiomer of the drugis known to be 28 times more therapeutically potent that its R(−)counterpart. Still another example of chiral pharmaceuticals is providedby the family of beta-blockers, the L-form of propranolol is known to be100 times more potent that the D-enantiomer.

Synthesis of chiral compounds by standard organic synthetic techniquesgenerally leads to a racemic mixture which, in the aggregate, may have arelatively low specific bioactivity since certain of the stereoisomersin the mixture are likely to be biologically or functionally inactive.As a result, larger quantities of the material must be used to obtain aneffective dose, and manufacturing costs are increased due to theco-production of stereochemically “incorrect” and hence, inactiveingredients.

In some instances, certain isomers may actually be deleterious ratherthan simply inert. For example, the D-enantiomer of thalidomide was asafe and effective sedative when prescribed for the control of morningsickness during pregnancy. However, its L-thalidomide counterpart wasdiscovered to be a potent mutagen.

Methods are available for stereoselective synthesis that generallyinvolve chemical synthesis and isolation steps that are lengthy, complexand costly. Moreover, a synthetic scheme capable of producing onespecific enantiomer cannot be applied in a general way to obtain otheroptically active compounds. What is needed is a generalized approach tothe resolution of racemic mixtures produced by ordinary chemicalreactions, and a number of approaches have been used.

A widely used approach has been the selective precipitation of desiredcompounds from racemic mixtures. See, for example, Yoshioka et al. [U.S.Pat. No. 3,879,451], Paven et al. [U.S. Pat. No. 4,257,976], Halmos[U.S. Pat. No. 4,151,198], and Kameswaran [U.S. Pat. No. 4,454,344].

The above procedures successfully resolved racemic mixtures becausetreatment of the mixtures with optically pure reagents produceddiastereomers which, unlike the initial racemic compounds, havedifferent physical properties. Thus, fractional crystallization or otherphysical means may be employed to separate diastereomeric compounds.

Separation of diastereomers can also be carried out by chromatography.For example, Pollock et al. [J. Gas Chromatogr. 3: 174 (1965)] haveresolved diastereomeric amino acids by gas chromatography. Mikes et al.[J. Chromatogr. 112:205 (1976)] have used liquid chromatography toresolve diastereomeric dipeptides. In most cases, the optically purereagents have been in the stationary phase during chromatographicseparation, but they may also be used in elutants. Hare et al. [U.S.Pat. No. 4,290,893] have used liquid chromatography to resolve racemicmixtures that were treated with aqueous elutants containing opticallypure reagents and metal cations; resolution occurred because theresulting diastereomeric complexes had different partition coefficientsin the chromatographic system.

All of the methods described to this point have relied upon theavailability of suitable optically pure reagents, but such reagents areoften not available or else their use is prohibitively expensive. In analternative approach, enzymatic resolution techniques have beendeveloped. Many different classes of enzymes have been used for theresolution of stereoisomers on a preparative scale, including hydrolases(especially the lipases and esterases such as chymotrypsin), lyases, andoxidoreductases (e.g., amino acid oxidases and alcohol reductases).Generally speaking, enzymes for use in resolutions should ideallyexhibit broad substrate specificity, so that they will be capable ofcatalyzing reactions of a wide range of “unnatural” substrates, and ahigh degree of stereoselectivity for catalyzing the reaction of oneisomer to the exclusion of others.

The hydrolases (e.g., lipases and esterases) are among the moreattractive enzymes for use in resolutions, because they do not requireexpensive cofactors, and some of them exhibit reasonable tolerance toorganic solvents. Additionally, chiral chemistry often involvesalcohols, carboxylic acids, esters, amides, and amines withchiral-carbons, and carboxyl hydrolases are preferred choices asstereoselective catalysts for reactions of such species. For instance,enzymatic treatment has been applied to the resolution of racemicmixtures of amino acid esters. Stauffer [U.S. Pat. No. 3,963,573] andBauer [U.S. Pat. No. 4,262,092].

Separation of reaction products from enzymes has been facilitated byattaching the enzyme to a solid support which could be removed bycentrifugation or packed into a column through which the racemicmixtures were passed.

Enzymes have also been explored for the resolution of classes ofcompounds other than the amino acids discussed above. Immobilized lipasein principal resolves mixtures by enzymatic hydrolysis ortransesterification. In the case of a biphasic hydrolysis reaction, thediffering solubility properties of the acids and esters involvedrequired the dispersion and agitation of mixtures containing theimmobilized solid-phase enzyme, an aqueous buffer, and thewater-immiscible organic phase containing solvent and reactant—arelatively inefficient process.

Enzymes have been applied to the resolution of optical isomers ofinsecticides. For instance, Mitsuda et al. [Eur. Patent Appl'n. Publ.No. 0 080 827 A2] contacted a racemic acetic acid ester withstereoselective esterases of microbial and animal origin in biphasicsystems (i.e., aqueous/organic dispersion). In related work on opticallypurified pyrethroids, Mitsuda et al. [U.S. Pat. No. 4,607,013] employedmicrobial esterases. Klibanov et al. [U.S. Pat. No. 4,601,987] resolvedracemic 2-halopropionic acids by means of lipase-catalyzedesterification reactions conducted in organic media.

Additional examples can also be provided of the state-of-the-artenzyme-mediated resolution as applied to the production of opticallypurified pharmaceuticals. Sih [U.S. Pat. No. 4,584,270] has disclosedenzymatic means for the production of optically pure(R)-4-amino-3-hydroxybutyric acid, a key intermediate in the preparationof L-carnitine.

Until recently only naturally occurring L-enzymes could be described,and this left the presumed properties of D-enzymes, including theirfolded structures, enzymatic activity and chiral specificity, asunexplored questions. What was needed was sufficient progress in thechemical synthesis of proteins to make possible the total synthesis ofthe D-enantiomer of whole enzymes in sufficient quantity to formcrystals and to perform other functions.

BRIEF SUMMARY OF THE INVENTION

A new type of enzyme designated a D-enzyme has been discovered that hasability to catalyze the reaction of a chiral substrate. Therefore,described herein is a D-enzyme comprising an amino acid residue sequencethat defines an polypeptide able to catalyze an enzymatic reaction,wherein the amino acid residue sequence consists essentially of D-aminoacids.

The enzymatic reaction can have achiral substrate specificity, or chiralsubstrate specificity. Preferred achiral substrate-specific D-enzymesare superoxide dismutase or carbonic anhydrase. A preferred chiralsubstrate-specific D-enzymes is HIV-1 protease.

The invention also contemplates a method of producing a chirally purechemical comprising:

-   -   a) reacting in an aqueous admixture a first stereoisomer        substrate with a D-enzyme that specifically converts the first        stereoisomer substrate into a chiral reaction product, wherein        the reaction occurs for a time period and under reaction        conditions sufficient to form a reaction product; and    -   b) isolating the chiral reaction product from the admixture,        thereby forming the chirally pure chemical. In alternative        embodiments, the aqueous admixture may comprise a racemic        mixture or partial racemic mixture of a substrate having at        least a first and a second stereoisomer or may comprise the        first stereoismer of the substrate alone.

A D-enzyme of this invention provides a wide variety of benefits andadvantages which are apparent to the skilled practitioner. A D-enzymeprovides a means to efficiently produce chirally pure chemicals for useas reagent grade industrial chemicals, and as pharmaceutically puremedicaments. In addition, a D-enzyme can be used in combination with itsL-enzyme counterpart in co-crystallation admixtures to form racemiccrystals for determining crystallographic structures using X-raydiffraction data. Furthermore, because of the inherent resistance of aD-enzyme from proteolysis by natural L-amino acid-specific proteases,therapeutically administered D-enzymes that have achiral substratespecificity can be utilized in place of the corresponding L-enzyme andenjoy prolonged half-lives in proteolytic environments such as the bloodor digestive tract, thereby increasing the effectiveness of thetherapeutic enzyme.

A D-enzyme of the present invention may also be employed for screeningnatural product libraries. More particularly, a D-enzyme may be employedto identify chiral inhibitors within a natural product library. In someinstance, a natural product libarary may include a chiral inhibitorhaving activity with respect to D-enzyme but having no activity withrespect to the corresponding L-enzyme. Synthesis of the enantiomer ofthe identified chiral inhibitor then results in the formation of aninhibitor of the corresponding L-enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates the molecular weight characterization of the D- andL-enzyme enantiomers of the HIV-1 protease as described in Example 2using reconstructed ion spray mass spectroscopy. The molecular weight isexpressed in daltons, and is shown as a peak of the percent (%) ofrelative intensity of the measured spectra. FIG. 1A illustratesmolecular weight data obtained with the L-enzyme, and FIG. 1Billustrates data obtained with the D-enzyme.

FIG. 2 illustrates the comparative enzyme activity of the HIV PR enzymeD- and L-enantiomers on D- and L-enantiomers of a chiral fluorogenicsubstrate as described in Example 3. FIG. 2A shows L-enzyme withL-substrate; FIG. 2B shows L-enzyme with D-substrate; FIG. 2C showsD-enzyme with L-substrate; and FIG. 2D shows D-enzyme with D-substrate.Data is expressed as activity, measured in arbitrary units offluorescence intensity, over a reaction time course in minutes.

FIG. 3 illustrates ribbon representations of the polypeptide backbone ofthe homodimeric HIV-1 protease molecule in both L- and D-conformations,shown in the left and right portions of the Figure, respectively. Thearrows indicate the direction of the polypeptide in the amino- tocarboxy-terminus direction.

FIG. 4 illustrates a schematic representation of the chemical segmentligation strategy employed for the total synthesis of D- andL-[Aba^(67,95) (CO—S)⁵¹⁻⁵²]₂ HIV-1 protease analogs.

FIG. 5 illustrates a schematic representation of the optimizedsolid-phase chain assembly tactics employed in the synthesis of thefunctionalized peptide segments. Deprotection and coupling reactions areseparated by a single flow wash step.

FIG. 6 illustrates a composite chromatogram showing two purifiedfunctionalized unprotected D-[αCOSH]HIV-1 PR(1–51) andD-[N^(α)-BrCH₂CO]HIV-1(53–99) segments and the final (48 hour)D-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 PR ligation product (bold) fun on aVydac 218TP5415 column eluted by gradient (40–55% B), at a flow rate of1 milliliter per minute.

FIG. 7 illustrates the step reaction yields for the synthesis of the D-and L-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 protease analogs.

FIG. 8 illustrates the ion spray mass spectra of the HPLC purified[(NHCH₂COSCH₂CO)^(51.52)HIV-1 PR monomers.

FIG. 9 illustrates the reverse phase HPLC measurements of D- &L-[Aba^(67,95), (CO—S)⁵¹⁻⁵²] HIV-1 PR ligation products.

FIG. 10 illustrates the far-ultraviolet circular dichroism spectrum ofthe D- and L-[(Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 protease analogs.

FIG. 11 illustrates the enzymatic activity of the [Aba^(67,95),(CO—S)⁵¹⁻⁵²]₂ HIV-1 PR enantiomers on D- and L-isomers of the substrateAc-Thr-Ile-Nle-Nle-Gln-Arg.amide.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are either in the “L” or “D” stereoisomericform. NH₂ refers to the free amino group present at the amino terminusof a polypeptide. COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. In keeping with standard polypeptidenomenclature (described in J. Biol. Chem., 243:3552–59 (1969) andadopted at 37 C.F.R. 1.822(b)(2)), abbreviations for amino acid residuesare shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine JXaa Unknown or other

The above symbols are employed for both L- and D-amino acid residues.The symbol Xaa is employed for any unknown or other amino acid residue.However, the symbol Xaa is frequently employed herein to designate L- orD-α-amino-n-butyric acid (aba), an isosteric replacement for Cysresidues. It should be noted that all amino acid residue sequencesrepresented herein by formulae have a left-to-right orientation in theconventional direction of amino terminus to carboxy terminus. Inaddition, the phrase “amino acid residue” is broadly defined to includethe amino acids listed in the Table of Correspondence and modified andunusual amino acids, such as those listed in 37 C.F.R. 1.822(b)(4), andincorporated herein by reference. Furthermore, it should be noted that adash at the beginning or end of an amino acid residue sequence indicatesa peptide bond to a further sequence of one or more amino acid residuesor a covalent bond to an amino-terminal group such as NH₂ or acetyl orto a carboxy-terminal group such as COOH.

Racemic Mixture: A racemic mixture is used herein to refer to a mixtureof at least a first and second stereoisomer in any proportions. In thiscontext, the term “resolution” as used herein will refer to separationof a first racemic mixture into second and third mixtures wherein theproportions of the two stereoisomers in the second and third mixturesare different from that in the first racemic mixture, the proportionbeing greater in one and necessarily smaller in the other.

B. D-Enzyme Compositions

The present invention contemplates a D-enzyme comprising a moleculehaving an amino acid residue sequence that defines a polypeptide able tocatalyze an enzymatic reaction. A D-enzyme has an amino acid residuesequence consisting essentially of D-amino acids.

The term “D-amino acid” does not indicate the direction of specificrotation of the molecule because it is well known that some amino acidsare dextrorotatory whereas others are levorotatory. Rather, the termsdenotes an absolute configuration by convention relative to the twopossible stereoisomers-of glyceraldehyde, D-glyceraldehyde andL-glyceraldehyde. See for example, Lehninger, in “Biochemistry”, WorthPublishers, Inc., New York, 1970, pp. 76–78. Thus all stereoisomers thatare stereochemically related to L-glyceraldehyde are designated L-, andthose related to D-glyceraldehyde are designated D-, regardless of thedirection of rotation of plane polarized light given by the isomer.

In the case of threonine and isoleucine, there are two stereochemicalcenters, i.e. the amino acid Cα atoms and the Cβ atoms. The D-threonineand D-isoleucine employed herein have stereochemistries at both theamino acid Cα atoms opposite to the stereochemistry of L-threonine andL-isoleucine, i.e. D-threonine and D-isoleucine are complete mirrorimages of L-threonine and L-isoleucine, respectively.

Glycine is the only commonly occurring achiral amino acid. Accordingly,when a protein or enzyme is designated herein as a D- or L-protein orenzyme, it is meant that essentially all of the chiral amino acidresidue comprising such protein or enzyme have the indicated chirality.The presence of achiral amino acid residues such as glycine within aprotein or enzyme does not affect the designation of its chirality, asemployed herein.

All chiral amino acids in protein described in nature are L-amino acids.Thus, proteins having only D-configuration chiral amino acids in theiramino acid residue sequence (referred to as D-proteins) are unknown innature.

In one embodiment, it is preferred that a D-enzyme have an amino acidresidue sequence that corresponds, and preferably is identical to, theamino acid residue sequence of a known or “natural” enzyme. By “natural”is meant a sequence present on an enzyme isolated from nature withoutlaboratory-mediated interventions directed at altering the enzyme'ssequence. By “known” is meant either a natural enzyme or an enzyme thatis the product of a sequence modifying process that alters the aminoacid sequence to produce an enzyme with known enzymatic properties.

Many enzymes described in the scientific literature, too numerous torecite here, have been the subject of mutation of their natural aminoacid residue sequence such that they no longer correspond in amino acidresidue sequence to the sequence of a natural isolate, and yet stillretain an enzymatic activity. Thus, in another embodiment, the inventioncontemplates D-enzymes having amino acid residue sequences thatcorrespond to known enzymes.

A D-enzyme can have any of a variety of enzymatic activities as thatactivity is generally understood in biochemistry, meaning broadly theability to reduce the activation energy of a reaction between one ormore substrates to form one or more reaction products. For the purposesof this invention, it is useful to distinguish enzyme substratespecificities that are chiral and achiral.

Chiral specificity refers to the selectivity of an enzyme to catalyzethe reaction of only one of two stereoisomers. Achiral specificityrefers to the ability of the enzyme to react with a substrate that doesnot present a recognition-dependent asymmetric structure to the enzyme,i.e., enzyme-substrate recognition and catalysis is not dependent uponthe presence of an asymmetric structure in the substrate binding regionof the enzyme. Stated differently and in the context of the presentinvention relating to enantiomeric selectivity of a D-enzyme, an achiralsubstrate can be catalyzed by either a D- or L-enzyme because noasymmetric structures are present in the achiral substrate upon whichenzyme binding and catalysis depends. In contrast, a chiral substratecan only be catalyzed by one or the other of a D- and L-enzyme pairbecause structural asymmetry of the substrate is involved in the bindingand catalysis.

A further distinction can be made between chiral and achiral reactionproducts. For example, an achiral substrate may be converted into achiral or an achiral reaction product. If an achiral substrate isconverted to a chiral reaction product, the chirality of the reactionproduct will depend upon the chirality of the enzyme, i.e. an L- orD-enzyme. Similarly, a chiral substrate may be converted into a chiralor an achiral reaction product.

Many enzymes exhibit chiral specificity including the preferred andexemplary enzyme, HIV-1 protease. Similarly, there are many enzymes thatexhibit achiral specificity, including superoxide dismutase and carbonicanhydrase.

Thus in one embodiment, the invention contemplates a D-enzyme havingchiral specificity that converts (catalyzes the reaction of) a chiralsubstrate into a reaction product, but does not also convert theenantiomer (stereoisomer) of the chiral substrate. An example is theHIV-1 protease described herein which reacts only with the D-substrateand not the L-substrate.

In another embodiment, the invention contemplates a D-enzyme havingachiral specificity wherein both the D-enzyme and the correspondingL-enzyme convert an achiral substrate into a reaction product. Oneexample is the reaction catalyzed by superoxide dismutase uponsuperoxide radicals. Another example is the reaction catalyzed bycarbonic anhydrase.

A D-enzyme of this invention can be any size (length of amino acids),and can be comprised of multiple subunits, as is well known for manycharacterized enzymes. A multiple subunit D-enzyme is comprised of allD-protein subunits. Protein subunits that make up an enzyme, or singleprotein subunit enzyme, range widely in size. Typical enzyme subunitsare from 80 to 500 amino acid residues in length, although shorter andlonger proteins are known, from about 50 amino acid residues to sizes inexcess of 4000 amino acid residues.

The present invention in one embodiment generally concerns the use ofD-enzymes in processes for the stereoselective synthesis or resolutionof racemic mixtures of chiral organic acids, alcohols, amines, esters,amides, nitriles, hydantoins, and other chiral compounds in which anenzyme is used that is capable of stereoselectively catalyzing areaction to convert one isomer of a chiral precursor to a chemicallydistinct optically active compound. Enzymes are well suited to the roleof stereoselective catalysis inasmuch as they contain asymmetric,catalytically active sites in which the molecule being synthesized orundergoing resolution may bind. Because these enzyme active sites arethemselves asymmetric, they permit two enantiomers of a given racemicsubstrate to be acted upon differentially, and they permit chiralproducts to be formed from achiral precursors.

For example, many enzymes exist that effectively catalyze the hydrolysisor condensation of ester and amide chemical functional groups. Many ofthese enzymes, but not all of them, belong to either one of two mainclasses of enzymes known as hydrolases or lyases as defined in theRecommendations of the Commission on Biochemical Nomenclature, Elsevier,Amsterdam, The Nomenclature and Classification of Enzymes (1972) p.17–22. The term E.C. followed by a series of numbers as used herein,provides the identification of an enzyme pursuant to the CommissionRecommendations.

Types of enzymes useful in the practice of the present inventioninclude, but are not limited to, enzymes that catalyze the followingcategories of reactions:

hydrolysis of esters to form acids and alcohols;

formation of esters (i.e., esterifications) from acids and alcohols;

transesterification, i.e., reaction of an ester with an alcohol or acidto form a different ester and a different alcohol or acid;

transaminations (e.g., reaction between an alpha-keto acid and an aminoacid);

hydrolysis of amides (including peptide bonds and N-acyl compounds) toform acids and amines;

formation of amides (including peptides) from acids and amines (or aminoacids);

hydrolysis of amino acid hydantoins to yield carbamoyl amino acids andamino acids; and

hydrolysis of nitriles to form the corresponding amides and carboxylicacids (and in particular, hydrolysis of amino nitriles to amino amidesand amino acids).

Specific examples of such enzymes include but are not limited totrypsin, chymotrypsin, thermolysin, rennin, pepsin, papain, carboxypeptidases, amino peptidases, penicillin and cephalosporin acylase,acetyl cholinesterase, cholesterol esterase, and mammalian pancreaticlipases and peptidases

Preferred esterases include chymotrypsin (E.C. 3.4.21.1) because of itshigh stereoselectivity, and broad substrate range. Other esterasesinclude, but are not limited to, carboxyl esterase (E.C. 3.1.1.1.),carboxypeptidase A (E.C. 3.4.17.1), acetyl cholinesterase (E.C.3.1.1.7), pepsin (E.C. 3.4.23.1), trypsin (E.C. 3.4.21.4) and papain(E.C. 3.4.22.2).

Amino acid residue sequences for natural enzymes, and published modifiedenzymes, useful for the present invention are generally available in thepublished literature and on computer data bases. Preferred and widelyused protein sequence data bases include Geneseq™, GenBank®, EMBL,Swiss-Prot, PIR and GenPept, all of which are commercially availablefrom Intelligenetics, Inc. (Mountain View, Calif.).

The complete three-dimensional structure for many enzymes suitable foruse in this invention are available from the Brookhaven Protein DataBank, Brookhaven National Laboratories, Upton, N.Y. Exemplary proteinswith their respective Protein Data Bank Codes (PDB numbers) that areincluded in the data base include:

-   (1hvp): hiv-1 protease complex with substrate;-   (2hvp): hiv-1 protease; (3hvp): (aba==67,95==)-hiv-1 protease, sf2    isolate; (4hvp): hiv-1 protease complex with the inhibitor    n-acetyl-*thr-*ile-*nle-psi(ch2-nh)-*nle-*gln-*arg amide (mvt-101)    (SEQ ID NO 1); (2cyp): cytochrome c peroxidase (e.c.1.11.1.5);-   (1gp1): glutathione peroxidase (e.c.1.11.1.9);-   (4cat): catalase (e.c.1.11.1.6);-   (7cat): catalase (e.c.1.11.1.6); (8cat): catalase (e.c.1.11.1.6);    and (2sod): cu,zn superoxide dismutase (e.c.1.15.1.1).

Particularly preferred are the antioxidant enzymes of the superoxidedismutase (SOD) class. Because of the wide distribution of SOD enzymesin aerobic organisms, many isolates of SOD have been reported in manyspecies. A recent literature search revealed descriptions of thesequence of 26 different SOD enzymes in mammals, non-mammals, bacteria,yeast and plants including human EC-SOD, [Hjalmarsson et al., Proc.Natl. Acad. Sci. USA, 84:6340–6344 (1987)]; human SOD [Sherman et al.,Natl. Acad. Sci. USA, 80:5465–5469], and Schneider et al., Cell,54:363–368 (1988); bovine SOD [Steinman et al., J. Biol. Chem.,249:7326–7338, (1974)]; equine SOD [Lerch et al., J. Biol. Chem.,256:11545–11551 (1981)]; murine SOD [Getzoff et al., Proteins: Struct.Func. Genet., 5:322–336 (1989)]; porcine SOD [Schinina et al., FEBSLett., 186:267–270 (1985)]; rabbit SOD [Reinecke et al., Biol. Chem.,369:715–725 (1988)]; ovine SOD [Schinina et al., FEBS Lett., 207:7–10(1986)]; rat SOD [Steffens et al., Z. Physiol. Chem., 367:1017–1024(1986)]; drosophila SOD [Nucleic Acids Res., 17:2133–2133 (1989)];xenopus SOD [Eur. J. Biochem. (1989)]; brucella SOD [Beck et al.,Biochemistry, 29:372–376 (1990)]; caulobacter SOD [Steinman et al., J.Bacteriol. (1988)]; neurospora SOD [Lerch, J. Biol. Chem., 260:9559–9566(1985)]; photobacterium SOD

[Steffens et al., Z. Physiol. Chem., 364:675–690 (1983)]; schistosomaSOD [Simorda et al., Exp. Parasitol., 67:73–84 (1988)]; yeast SOD[Steinman et al., J. Biol. Chem., 255:6758–6765 (1980)]; cauliflower SOD[Steffens et al., Biol. Chem. Hoppe-Seyler, 367:1007–1016 (1986)];cabbage SOD [Steffens et al., Physiol. Chem., 367:1007–1016 (1986)];maize SOD [Cannon et al., Proc. Natl. Acad. Sci. USA, 84:179–183(1987)]; pea SOD [Scioli et al., Proc. Natl. Acad. Sci. USA,85:7661–7665 (1988)]; spinach SOD [Kitagawa et al., J. Biochem.,99:1289–1298 (1986)]; and tomato SOD [Plant Mol. Biol., 11:609–623(1988)]. Any of these varieties of SOD are suitable for use as aD-enzyme of the present invention.

Carbonic anhydrase C is another preferred enzyme suitable for thepreparation of a D-enzyme. Carbonic anhydrase C catalyses the reactionthat combines carbon dioxide and water to form bicarbonate and hydrogenions. The sequence of carbonic anhydrase C is described by Henderson etal., Biochim. Biophys. Res. Comm., 52:1388 (1973); Lin et al., J. Biol.Chem., 249:2329 (1974).

Other optically specific enzymes that react with a chiral substrate andare therefore useful as a D-enzyme of this invention have beenextensively described in U.S. Pat. Nos. 5,077,217, 5,057,427, 4,800,162,and 4,795,704, which are specifically incorporated herein by reference.

C. Synthesis of a D-Enzyme

A D-enzyme of the present invention can be prepared by any meansavailable to one skilled in the polypeptide arts. The precise methodemployed for synthesizing the polypeptide is not considered essential tothe basic structure of a D-enzyme of this invention, and therefore isnot to be considered as limiting, particularly as technology developsnew ways to synthesize and assemble polypeptides.

Preferred routes of polypeptide synthesis include:

-   -   1. Conventional chemical synthesis, e.g. step wise synthesis,        and    -   2. Assembly of polypeptide building blocks by chemical ligation.

It is presently considered impractical to employ conventional chemical(step-wise) synthetic methods to produce polypeptides having more than100 amino acid residues. On the other hand, chemical ligation methodsmay be employed to assemble polypeptides several time larger.Accordingly, for D-enzymes greater than 100 amino acid residues,chemical or enzymatic ligation techniques are presently the onlypractical means for making such products. Described herein is a ligationstrategy for preparing 10–100 milligram amounts of the D- and L-HIV-1protease enzymes.

Although not presently available, manufactured protein synthesisapparati using D-proteins may solve the problem of incorporating D-aminoacids in the protein translation machinery, making it possible tosynthesis D-enzymes using recombinant DNA expression of messenger RNAand D-amino acids.

Conventional Step-wise Syntheses:

Synthetic chemistry techniques, such as the stepwise addition of aminoacids in a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, ease of production and the like. An excellent summary of themany techniques available for synthesizing L-proteins and enzymes can befound in Steward et al., in “Solid Phase Peptide Synthesis”, W.H.Freeman Co., San Francisco, 1969; Bodanszky et al., in “PeptideSynthesis”, John Wiley & Sons, Second Edition, 1976 and Meienhofer, in“Hormonal Proteins and Peptides”, Vol. 2, p. 46, Academic Press (NewYork), 1983; and Kent, Ann. Rev. Biochem., 57:957, 1988, for solid phasepeptide synthesis, and Schroder et al., in “The Peptides”, Vol. 1,Academic Press (New York), 1965 for classical solution synthesis, eachof which is incorporated herein by reference. Appropriate protectivegroups usable in such synthesis are described in the above texts and byMcOmie, in “Protective Groups in Organic Chemistry”, Plenum Press, NewYork, 1973, which is incorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

For the synthesis of a D-enzyme, D-amino acids or protected D-aminoacids are utilized rather than the conventional L-amino acids. D-aminoacids suitable for polypeptide synthesis are commercially available fromthe Peptide Institute (Osaka, Japan); Peptides International(Louisville, Ky.); Bachem Bioscience (Philadelphia, Pa.); and BachemCalifornia, (Torrance, Calif.).

Using a solid phase synthesis as exemplary, the protected or derivatizedD-amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next D-amino acidin the sequence having the complimentary (amino or carboxyl) groupsuitably protected is admixed and reacted under conditions suitable forforming the amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added D-amino acid residue, and the next D-aminoacid (suitably protected) is then added, and so forth. After all thedesired D-amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

Ligation Techniques:

The chemical ligation of polypeptides has recently been described byKent in U.S. application Ser. No. 07/865,368, filed Apr. 8, 1992, whosedisclosures are hereby incorporated by reference. This technique ispreferred for D-enzymes having a length of 100 amino acid residues orgreater. In this procedure, two polypeptides are first synthesized, andcontain termini adapted for chemical ligation. After stepwise chemicalsynthesis and cleavage from their respective solid phase resins, the twopolypeptides are mixed and reacted to join the adapted termini and forma larger, linear polypeptide comprised of the two polypeptides.

An exemplary step-wise synthesis of a D-enzyme is detailed in Example 1describing the synthesis of a variety of D-HIV-1 protease. Example 5discloses an exemplary ligation-type synthesis of D- andL-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 protease analogs. TheD-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 protease analog of Example 5 isfunctionally equivalent to the D-HIV-1 protease of Example 1.

Similar synthesis can be applied to the preparation of D-superoxidedismutase, carbonic anhydrase, or any of the other D-enzymes describedherein.

D. Methods for Screen Chemical Libraries

The most common means for identifying pharmaceutically useful compoundsinvolves screening chemical libraries. The thoroughness such screeningmay be markedly enhanced by employing both L-enzymes and D-enzymes.

Natural product libraries isolated from nature may consist of severalhundred thousand compounds. Chemical libraries may also be prepared bychiral synthesis of particular enantiomers or by non-chiral synthesis ofracemates. In the search for new drug candidates, such libraries may bescreened to identify compounds active in a particular assay. In manyinstances, the target molecule is an enzyme or an enzyme system and thesearch is directed to identifying drug candidates which can serve asspecific inhibitors or cofactors of such enzyme or enzyme system. Once acandidate compound is identified as an inhibitor of a specifictherapeutically relevant enzyme, analogues of such candidate compoundmay be designed and synthesized so to improve its activity and otherdesireable properties.

In many instances, chiral specificity is a necessary attribute of activesubstrates, cofactor, and inhibitors. However, it is disclosed hereinthat the chiral specificity of substrates, cofactors, and inhibitorsdepends upon the chirality of the target enzyme. Accordingly, the targetenzyme can often distinguish between active and inactive enantiomers ofa given a substrate, cofactor, and inhibitor. Component elements of anatural product library often display random chirality and bear toinherent relationship to the target enzyme. Accordingly, if only asingle enantiomer is present within a library, the chirality of suchenantiomer is as likely to be the wrong (inactive) enantiomer as to bethe right (active) with respect to any given target enzyme. If thelibrary is screened against only the native (L)-configuration of atarget enzyme, and if the elements of the library are non-racemate, i.e.if they are chiral, there is a significant chance (50/50) that thelibrary include only the inactive enantiomer.

The present invention teaches that screening a natural product libraryagainst both an L-enzyme and its corresponding D-enzyme, canapproximately double the number of candidate compounds identified fromsuch library.

Any candidate compound identified as active against a D-enzyme may beinactive with respect to the corresponding L-enzyme. However, structuralanalysis of a candidate compound active with respect to a D-enzyme ispredictive of the structure of a candidate compound active with respectto the corresponding L-enzyme, i.e. the enantiomers of compounds foundto be active with respect to a D-enzyme are likely to have correspondingactivity with respect to the corresponding L-enzyme.

Accordingly, the number of candidate compounds positively identifiedfrom a chemical or natural product library may be significantly enhancedif the library is screened against both the L- and D-version of theenzyme. For example, screening a natural product library with respect tothe inhibition of the protease activity of both L- and D-HIV proteaseshould significantly increase the number candidate drugs identified asactive inhibitors.

The above concepts apply equally to screening natural product librarieswith respect to receptor activity, i.e. as agonist or antagonist withrespect to protein receptors. Included amongst protein receptors againstwhich natural product and/or chemical libraries may be usefully screenedare the following: GPIIb-IIIa and LFA-1, Ruoslahti et al., Science, 238:491–497 (1987); CSAT, Horwitz et al., J. Cell Biol., 101:2134 (1985);VLA-2, Nieuwenhuis et al. Nature, 318:470 (1985); CR3 ComplementReceptor, Wright et al., PNAS, 84:1965 (1987); CR2 Complement Receptor,Nemerow et al., J. Virol., 55: 3476 (1985); CD4 T Cell Receptor, Guyaderet al., Nature, 320:662 (1987); FRP Receptor, Yu et al., Nature, 330:765(1987); Apolipoprotein Receptor, Yamada et al., J. Clin. Invest., 80:507 (1987); Interleukin Receptor, Dower et al., Immunology Today, 8:46(1987); Fc Receptor, Anderson et al., J. Immunol., 138: 2254 (1987);Somatostatin Receptor, Kim et al., J. Biol. Chem., 262: 470 (1987); PDGFReceptor, Keating et al., J. Biol. Chem., 252: 7932 (1987); andTransferrin Receptor, Kohgo et al., Blood, 70:1955 (1987).

Other proteins having binding sites which may be screened according tothe method of the present invention include insulin receptor bindingsite on insulin, reovirus receptor binding site on the firalhemaglutinin protein, fibrinogen receptor binding site on figrinogen Aalpha, thyroid hormone receptor binding sites α and β, LDL receptorbinding site on Apo E, lipid A binding site, lecithin-cholesterolacyltransferase (LCAT) binding site on Apo AI, and Mac-1 integrinreceptor binding site on fibrinogen D-30.

E. Methods for Producing Chirally Pure Compounds

In another embodiment, the present invention contemplates methods usinga D-enzyme of this invention for the production of chirally purechemical compounds. A chirally pure compound, as used herein refers to amolecule substantially free from its stereoisomer.

The methods can be practiced in a variety of ways. A single chirallypure chemical can be produced by a reaction by D-enzyme upon a racemicmixture of substrates, leaving in the racemic mixture one of thesubstrate isomers, and converting the other substrate isomer into aproduct. In this method, the desired chirally pure compound can be areaction product, or it can be the substrate isomer left unreacted inthe racemic mixture, freed from the contaminating isomer by the actionof the D-enzyme.

Thus, in this embodiment, the invention contemplates a method ofproducing a chirally pure chemical comprising:

-   -   a) reacting in an aqueous admixture a first stereoisomer with a        D-enzyme that specifically converts said first stereoisomer into        a chiral reaction product; and    -   b) isolating the chiral reaction product from the admixture,        thereby forming the chirally pure chemical. In an alternative        version of this embodiment, the aqueous admixture comprises a        racemic mixture having at least a first and a second        stereoisomer.

The reaction is initiated by admixing D-enzyme with substrate andsubjecting the reaction admixture to suitable reaction conditions fordriving the enzyme catalyzed reaction. For a D-enzyme those conditionsdepend upon the particular reaction chemistry to be catalyzed and uponthe conditions under which the enzyme is active. The reaction conditionsfor a D-enzyme are preferably the same as is optimally used for thecorresponding reaction of the isomeric substrate by the correspondingL-enzyme. Preferred reaction conditions are those temperature andaqueous buffer conditions which favor maximum enzyme activity for thedesired reaction and minimum undesirable side reactions.

The isolating step can be conducted by any chemical manipulation thatprovides for the resolution of the chirally pure chemical from thereaction product admixture formed in step (a). Exemplary isolatingmanipulations are well know to the chemist and include solventextractions, chromatography, selective crystallization, distillation,and the like.

In a related embodiment, the invention contemplates a method forproducing a chirally pure chemical comprising:

-   -   a) reacting in an aqueous admixture a racemic mixture having at        least a first and a second stereoisomer with a D-enzyme that        specifically converts the first stereoisomer into a reaction        product; and    -   b) isolating said second stereoisomer from said admixture,        thereby forming said chirally pure chemical. The reaction is        conducted for a time period and under reaction conditions        sufficient to convert substantially all of the first        stereoisomer into the chiral reaction product.

In this latter embodiment, depletion of the original racemic mixture ofan undesirable stereoisomer resolves the chirally pure chemical, and theremaining unreacted stereoisomer is isolated to form the chirally purechemical.

Chemical synthesis of a chirally pure chemical using a D-enzyme can beconducted in a homogeneous or heterogeneous aqueous reactionenvironment, or can be conducted in enzyme reactors, where the D-enzymeis in the solid phase, or in membrane reactors, where a solvent orD-enzyme is segregated away from either the reactants or products. Suchsolid phase enzyme reactors and membrane enzyme reactors, and theirmethods of use, have been extensively described in U.S. Pat. Nos.5,077,217, 5,057,427, 4,800,162, and 4,795,704, which are specificallyincorporated herein by reference.

F. Methods of Co-Crystallization Racemic Mixtures

In one embodiment, the invention contemplates the use of a D-enzyme toproduce an X-diffraction pattern of a crystal for determining the threedimensional structure of a protein. Methods for preparing crystallizedproteins and analyzing the crystal structure by the X-raycrystallographic arts is well known. See for example the teaching ofStout et al., in “X-Ray Structure Determination: A Practical Guide”,Macmillan, N.Y., 1968; and Miller et al, J. Mol. Biol., 204:211–212,1988, which are hereby incorporated by reference.

In a preferred embodiment, the invention contemplates theco-crystallization of the D- and L-HIV-1 protease enzymes prepared asdescribed in Example 1. The resulting crystal, formed by the vapordiffusion crystal growth method described by Miller et al., supra, isused to solve the three dimensional structure of HIV-1 protease usingconventional X-ray diffraction methods to produce a racemic crystal dueto the presence of the enantiomeric forms (D- and L-) of HIV-1 proteasein the crystal.

Such a crystal structure is further useful, for example, to modelinhibitors of HIV-1 protease useful for therapeutic treatment of HIV-1infection by inhibition of the protease.

Co-crystallization of D- & L-HIV protease preparations can generatecentrosymmetric crystals for high-accuracy X-ray diffraction studies.This should provide data that can find a wide usefulness in drug designstudies for AIDS therapeutics. For this purpose, it is necessary toproduce each enantiomorph of the enzyme in hundred milligram quantitieswithout the complication of autolysis during the extraction,purification and folding of the synthetic products. We have disclosedthat an HIV-1 protease analogue, prepared by the directed chemicalligation of unprotected peptide segments and containing a thioesterreplacement for the natural peptide bond between Gly⁵¹-Gly⁵², has fullactivity. This segment condensation strategy also largely avoids thepossibility of autodigestion by the enzymes during their preparation.

G. Therapeutic Methods

The invention contemplates therapeutic methods involving administrationof therapeutically effective amounts of a D-enzyme to a mammal or human,where an L-enzyme would normally be the active ingredient in thetherapeutic composition to be administered. By substituting a D-enzymefor its corresponding L-enzyme, the therapeutic enzyme acquires thebenefits of a D-enzyme as described herein, including increasedeffective half-life due to resistance to proteases, and diminishedimmune recognition.

Enzymes for use in therapeutic treatment methods as a D-enzyme of thisinvention can be derived from any number of enzymes of known primaryamino acid residue sequence that provide therapeutic applications, andwhich have achiral substrates. Antioxidant enzymes, in particular, havetherapeutic applications that can benefit from being in the form of aD-enzyme to increase effective therapeutic half-life.

Antioxidants function as anti-inflammatory agents. The medicallyimportant antioxidant enzymes of known structures are superoxidedismutase, catalase and glutathione peroxidase. These enzymes areinvolved in the prevention of post-ischemic injuries and the control ofinflammatory disorders. Wilsman et al., In: Superoxide and SuperoxideDismutase in Chemistry, Biology and Medicine. Rotilio, Ed., ElsevierScience, Amsterdam Publishers (1986). As demonstrated in the case ofSOD, these enzymes would benefit from an increased circulatoryhalf-life.

1. Superoxide Dismutase

The SOD enzymes, which catalyze the conversion of superoxide radical tomolecular oxygen and hydrogen peroxide, are ubiquitous in organisms thatutilize oxygen.

The dismutation reaction of SOD enzymes is important in preventingtissue damage by free radicals. Indeed, the effectiveness of humanintracellular SOD (HSOD) in relieving inflammatory disorders includingosteoarthritis has been demonstrated by clinical studies in humans. See,Wilsmann, Superoxide and Superoxide Dismutase in Chemistry, Biology andMedicine, Elsevier, 500–5 (1986). Additionally, animal studies havesuggested that SOD enzymes have therapeutic potential for viralinfections. See, Oda et al., Science, 244:974–6 (1989). SOD enzymes havealso been implicated in preventing alloxan diabetes [Grankvist et al.,Nature, 294:158 (1981)] and in preventing metastasis of certain forms ofcancer (EPO Application No. 0332464).

Therefore in one embodiment, methods for reducing tissue damage causedby oxygen free radical (superoxide) in vivo or in vitro are contemplatedby the present invention, using a D-superoxide dismutase (D-SOD) enzymeof this invention.

Human recombinant SOD can protect ischemic tissue in experimental modelswhen injected into the circulation just prior to reperfusion (Ambrosioet al. Circulation 75:282. 1987). Injury to the endothelium, a tissuecovered with glycosaminoglycans, is a major consequence ofischemia/reperfusion injury. This causes edema formation due to the lossof barrier function and favors platelet adhesion to endothelium. Theprotective action of SOD is due to its scavenging of superoxide anion.SOD can also protect the endothelium “in vivo” by preventing theformation of peroxynitrite, which is toxic due to its decomposition toform potent, cytotoxic oxidants (Beckman et al. Proc. Natl. Acad. Sci.USA 87:1620–1624. 1990). Postischemic injury involving the superoxideanion has been observed in the heart, intestine, liver, pancreas, skin,skeletal muscle, kidney and perhaps occurs in other organs (McCord Fed.Proc. 46:2402–2406. 1987). SOD chemically linked or conjugated toalbumin exhibits an increased “in vivo” half-life, i.e. slowerclearance. Such conjugated SOD has been shown to be superior tounconjugated SOD with respect to inhibiting postischemic reperfusionarrhythmias (Watanabe et al. Biochem. Pharmacol. 38:3477–3483. 1989).The preparation of D-SOD having a half-life greater than the half-lifeof L-SOD facilitates the use of SOD for preventing or diminishingpostischemic damage.

SOD has also proven to be effective in several inflammatory diseaseslike osteoarthritis and rheumatoid arthritis. Local infiltration of SODin extra-articular inflammatory processes (e.g., tendinitis,tendovaginitis, bursitis, epicondylitis, periarthritis) has also provento be effective. Improvement upon SOD administration has also beenobserved in Peyronie's disease and Dupuytren's contracture (Wilsman. InRotilio Ed. Superoxide and Superoxide Dismutase in Chemistry, Biologyand Medicine. Elsevier. 1986). For these inflammatory disorders as wellas for respiratory distress syndromes a cell surface targeted SOD withincreased half-life will be a useful drug. Organ transport and organtransplant also can benefit from such an improved SOD. In addition,tissue targeted SOD should help alleviate the toxic secondary effect ofanti-cancer radio- and chemotherapy. Drug (antibiotic and anticancer)induced nephritis also can be reduced by a more potent SOD. D-SOD may besubstituted to advantage for L-SOD in the above-recited therapeuticapplications.

Thus, the present invention contemplates a method of in vivo scavengingsuperoxide radicals in a mammal that comprises administering atherapeutically effective amount of a physiologically tolerablecomposition containing a D-SOD enzyme to a mammal in a predeterminedamount calculated to achieve the desired effect.

For instance, when used as an agent for scavenging superoxide radicals,such as in a human patient displaying the symptoms of inflammationinduced tissue damage such as during an autoimmune disease,osteoarthritis and the like, or during a reperfusion procedure toreintroduce blood or plasma into ischemic tissue such as during or aftersurgical procedures, trauma, in thrombi, or in transplant organs, orafter episodes of infection causing massive cell death and release ofoxidants, the D-SOD enzyme is administered in an amount sufficient todeliver 1 to 50 milligrams (mg), preferably about 5 to 20 mg, per humanadult, when the D-SOD enzyme has a specific activity of about 3000 U permg. A preferred dosage can alternatively be stated as an amountsufficient to achieve a plasma concentration of from about 0.1 ug/ml toabout 100 ug/ml, preferably from about 1.0 ug/ml to about 50 ug/ml, morepreferably at least about 2 ug/ml and usually 5 to 10 ug/ml.

D-SOD enzymes having superoxide dismutase (SOD) activity for use in atherapeutic composition typically have about 200 to 5000 units (U) ofenzyme activity per mg of protein. Enzyme assays for SOD activity arewell known, and a preferred assay to standardize the SOD activity in aD-enzyme is that described by McCord et al., J. Biol. Chem., 244:6049(1969).

For treating arthritic conditions such as rheumatoid arthritis,tendinitis, bursitis or the like, a dosage of about 1 to 20 mg,preferably about 4 to 8 mg is administered intra articularly per weekper human adult. In certain cases, as much as 20 mg can be administeredper kilogram (kg) of patient body weight.

For treating reperfusions, or myocardial injuries, a dosage of 5 mg perkg of body weight is preferred to be administered intravenously.

The therapeutic compositions containing a D-SOD enzyme areconventionally administered intravenously, or intra articularly (ia) inthe case of arthritis, as by injection of a unit dose, for example. Theterm “unit dose” when used in reference to a therapeutic composition ofthe present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or, more hour intervals by asubsequent injection or other administration. Alternatively, continuousintravenous infusion sufficient to maintain concentrations in the bloodin the ranges specified for in vivo therapies are contemplated.

Additional exemplary therapeutic applications of SOD, which are directlyapplicable to the present methods of using therapeutic D-SOD, andcompositions containing SOD useful therefor, are described in U.S. Pat.Nos. 5,084,390, 5,006,333 and 4,656,034, which disclosures arespecifically incorporated herein by reference.

Similarly to the improved therapeutic methods described above withD-SOD, many otherwise satisfactory enzyme pharmaceutical agents areexpected to find limited therapeutic use due to their short lifetimes invivo. Thus, a convenient method for extending the useful lifetimes ofproposed pharmaceutical agents is desired and is provided by thepreparation of a D-enzyme according to the present invention. Themethods herein allow the preparation of the D-enzyme variants of theenzyme pharmaceutical agents that at least have biological activitiescomparable to those for the unaltered agent.

H. Therapeutic Compositions

Many of the compounds and groups involved in the instant specification(e.g., D-amino acid residues) have a number of forms, particularlyvariably protonated forms, in equilibrium with each other. As theskilled practitioner will understand, representation herein of one formof a compound or group is intended to include all forms thereof that arein equilibrium with each other.

In the present specification, “uM” means micromolar, “ul” meansmicroliter, and “ug” means microgram.

Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with a D-enzyme, as describedherein, dissolved or dispersed therein as an active ingredient.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like and combinations thereof. In addition, if desired,the composition can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an amount of a D-enzyme of thepresent invention sufficient to deliver a catalytic amount of the enzymeto the target tissue to be treated. Typically this is an amount of atleast 0.1 weight percent, and more preferably is at least 1 weightpercent, of D-enzyme per weight of total therapeutic composition. Aweight percent is a ratio by weight of protein to total composition.Thus, for example, 0.1 weight percent is 0.1 grams of D-enzyme per 100grams of total composition.

EXAMPLES

The following examples are intended to illustrate, but not limit, thescope of the invention.

1. Conventional Step-wise Synthesis of L- andD-[Aba^(67,95,167,195)]HIV-1 Protease (HIV-1 PR)

Advances in the total chemical synthesis of proteins made possible thereproducible production of homogeneous crystallineL-[Aba^(67,95,167,195)]HIV-1 protease (HIV-1 PR). See, for example Kent,Annu. Rev. Biochem., 57:957, (1988); Wlodawer et al., Science, 245:616(1989); and Miller et al., Science, 246;1149 (1989).

HIV-1 protease (HIV-1 PR) is a virally-encoded enzyme which cutspolypeptide chains with high specificity and which is essential for thereplication of active virions. Kohl et al., Proc. Natl. Acad. Sci.U.S.A., 85:4686, 1988. The 21,500 dalton HIV-PR molecule is made up oftwo identical 99 amino acid polypeptide chains.

The total chemical synthesis of D-[Aba^(67,95,167,195)]HIV-1 proteasewas carried out as described below, and the properties (covalentstructure, physical properties, circular dichroism, enzymatic activity)of the D- and L-enantiomeric forms of this HIV-1 protease enzyme werecompared.

To that end, in separate chemical syntheses, the protected polypeptidechains corresponding to the L- and the D-sequences of the [Aba^(67,95)]HIV-1 protease 99-aa monomer were prepared by total chemical synthesis.Aba is L- or D-a-amino-n-butyric acid and is used as an isostericreplacement for Cys residues at positions 67 and 95 in the HIV PRmonomer polypeptide chain. This same isosteric replacement was used inthe work of Wlodawer et al., supra, and Miller et al., supra, leading tothe original correct structures of HIV PR.

The chemical synthesis was conducted in conventional stepwise fashion.The 99-aa polypeptide chains were assembled from protected L-amino acidsand protected D-amino acids, respectively. The t-Boc D- and L-amino acidderivatives were obtained from the Peptide Institute (Osaka, Japan) andPeptides International (Louisville, Ky.) except: Boc-L-Aba,Boc-L-Asn(Xan), Boc-D-Ile and Boc-D-His(Bom), obtained from BachemBioscience (Philadelphia, Pa.); Boc-D-Asn(Xan), Boc-D-Asp(OcHex) andBoc-D-Glu(OcHex), obtained from Bachem California, (Torrance, Calif.);Boc-D-Lys(ClZ), crystallized from the TBA salt obtained from the PeptideInstitute; and, D-Aba (Sigma, St. Louis, Mo.) which was converted toBoc-D-Aba and isolated as the DCHA salt. Other side chain protectinggroups that were used were: Arg(Tos), Tyr(BrZ), L-His(Tos), D-His(Bom)and Thr(BzL). The L-enantiomer content of the Boc-D-amino acidpreparations was between 0.01 and 0.08% [manufacturers specifications].Stepwise chain assembly was carried out in machine assisted fashion onan Applied Biosystems 430A synthesizer (0.2 mmole scale with D- orL-Boc-Phe-OCH₂-Pam-resin). Each cycle of amino acid addition involved:

-   N^(α)-deprotection, neat (100%) TFA [2×30 sec flow washes, plus 1    minute batchwise treatment]; DMF flow wash [1×22 sec, 1×38 sec];    coupling [1×10 minute] with simultaneous in situ neutralization    [Boc-amino acid (2.25 mmol) preactivated by reaction with HBTU (2.22    mmol) and DIEA (6.4 mmol) in DMF for 2 min]. The in situ    neutralization method has been shown to result in negligible levels    of racemization. Henklein et al., in “Innovation & Perspectives in    Solid Phase Synthesis”, R. Epton Ed., SPPC Ltd., Birmingham,    U.K., 1992. The assembled peptides were deprotected and cleaved from    the resin in 9:1 HF/p-cresol (resorcinol and thiocresol were present    when His(Bom) was included in the sequence) after removal of the Boc    group and the formyl group from Trp (with ethanolamine).

The D- and L-products after deprotection were worked up individually,and synthetic enzymes were then prepared by folding the polypeptidepolymers from denaturant as described by Wlodawer et al., supra, andMiller et al., supra. To that end, after deprotection and cleavage, thecrude peptide products were precipitated with ether and dissolved with6M guanidine hydrochloride in a pH 8.0 NaHCO₃ buffer prior tosemi-preparative C18 RP HPLC enrichment and folding by dialysis in 10%glycerol, 25 mM NaH₂PO₄ buffer pH 7.0. After concentration under highvacuum to a solution in glycerol, the enzymes were quantitated by aminoacid analysis and stored at 4° C. Total yield for the synthesis ofL-[Aba^(67,95,167,195)] HIV-1 Protease (HIV-1 PR) was approximately 2milligrams or 0.09%.

Total yield for the synthesis of D-[Aba^(67,95,167,195)]HIV-1 Protease(HIV-1 PR) was indeterminant because it was less than 2 milligrams.

2. Structural Analysis of the Above L- and D-[Aba^(67,95,167,195)]HIV-1Protease (HIV-1 PR)

The D-enzyme 99-aa monomer, D-[Aba^(67,95)] HIV-1 protease, was analyzedfor various structural characteristics, and compared to the structuralcharacteristics of the L-isomer. For example, analytical reversed-phaseHPLC gave identical retention times for the two synthetic polypeptidechains.

Purified, folded chemically synthesized [Aba^(67,95)]HIV-1 proteasemonomer samples prepared in Example 1 in pH 6.5 MES buffer/10% glycerolwere subjected to desalting by reverse phase HPLC. The collected proteinpeaks were each separately analyzed by ion spray mass spectrometry asdescribed by Bruins et al., Anal. Chem., 59:2642 (1987). Under theconditions used (50% acetonitrile, 50% water, 0.1% TFA) the enzyme isdenatured. In the reconstructed mass spectra shown, the raw m/z datahave been subjected to a high pass digital filter, then sorted to yieldall parent molecular species between 10 kDa and 11 kDa. Thisreconstruction procedure mathematically reduces the multiple chargestates observed for a given molecular species to a single molecularmass.

By reconstructed ion spray mass spectroscopy, the observed monomermolecular mass of the L-enzyme was 10,748±4 daltons (Da), and the massof the D-enzyme was 10,75±3 Da. Calculated mass: (monoisotopic) 10,748.0Da; (average) 10,754.7 Da.

Thus, the two products (D- and L-enzyme monomers) had the same molecularweight, within experimental uncertainty, when measured by ion spray massspectrometry. The ion spray mass spectrometry data are shown in FIG. 1.

The complete amino acid sequence of the D-enzyme 99-aa monomer wasdetermined by matrix-assisted laser desorption time-of-flight massspectrometric readout (Model API-III mass spectrometer, P.E. SCIEX Inc.,Thornhill, Toronto, Canada) and was shown to be the same as that of theL-enzyme. Thus, the two synthetic enzyme molecules had identicalcovalent structure.

On the other hand, differences between the two molecules were revealedin various chiral interactions. Circular dichroism (CD) spectra of theindividual D- and L-HIV-1 protease enantiomers were taken over the range260–195 nm in a pH 5.5 aqueous solution containing 5% glycerol at 25° C.using a 1 mm path length quartz cell on an Aviv CD spectrometer. The CDspectra revealed equal and opposite optical rotations, as expected forenantiomeric protein molecules.

3. Enzymatic Properties of the Above L- and D-[Aba^(67,95,167,195)]HIV-1Protease (HIV-1 PR)

The enzymatic properties of the enantiomeric protein comprised ofD-amino acids was evaluated and compared to the L-isomer using afluorogenic assay which employed a hexapeptide analog of a natural GAGcleavage site as substrate as described by Toth et al., Int. J. PeptideProtein Res., 36:544 (1990).

The fluorogenic assays were performed with 15 ul aliquots (correspondingto 1.75 (±10%) ug protein) of each enzyme enantiomer in 10% glycerol,100 mM MES buffer pH 6.5 added to a solution of 50 mM D- orL-fluorogenic substrate in the MES buffer. The substrate for the enzymehad the sequence 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg.amide.(SEQ ID NO 2). The substrate was synthesized with either D- or L-aminoacid derivatives to provide the appropriate enantiomeric forms.

The results of the fluorogenic assay are shown in FIG. 2. Aliquotscontaining equal amounts (as determined by amino acid analysis) of thepurified, folded enzyme preparations were used in the fluorogenic assay.The increase in fluorescence was recorded on a continuous chartrecorder. The data illustrate that the two synthetic enzyme moleculeswere equally active, but revealed a reciprocal chiral specificity inthat the L-enzyme cleaved only the L-substrate while the D-enzymecleaved only the corresponding D-substrate.

In a similar study, the D- and L-enantiomers of the pseudopeptideinhibitor, MVT101

(Ac-Thr-Ile-Nle-psi[CH₂NH]-Nle-Gln-Arg.amide), (SEQ ID NO 1) prepared asdescribed by Miller et al., Science, 246:1149 (1989), were evaluated fortheir effect on the D- and L-HIV protease. The results of the studiesusing inhibitor are shown in Table 1.

TABLE 1 Chiral inhibitors show reciprocal chiral specificity against D-and L-HIV PR^(a). L-MVT101 D-MVT101 Evans Blue^(b) L-HIV PR + − + D-HIVPR − + + ^(a)The D- and L-enzymes were separately assayed by thefluorogenic assay method described above using the corresponding chiralsubstrate, in the presence of 5xIC50 concentration of inhibitor. ^(b)Theinhibitor Evans Blue is a non-peptide, achiral mixedcompetitive-uncompetitive inhibitor of the HIV-1 PR.

The chiral inhibitors were effective only against the correspondingenantiomer of the enzyme, i.e. L-MVT101 inhibited L-HIV PR but not theD-HIV PR-catalyzed reaction, and D-MVT101 inhibited D-HIV PR but had noeffect on the L-enzyme-catalyzed reaction. Interestingly, the achiralinhibitor Evans Blue, which shows mixed inhibition kinetics, was apotent inhibitor of both enantiomers of the enzyme (Table 1).

The HIV-1 protease exists as a homodimer; that is, a single enzymemolecule is made up of two identical 99 residue folded polypeptidechains. Wlodawer et al., Science, 245:616 (1989); and Miller et al.,Science, 246;1149 (1989). HIV PR is highly active, showing rateenhancement of about 10¹⁰-fold over uncatalyzed peptide-bond hydrolysis.Kent et al., in “Viral Proteinases as Therapeutic Targets”, Wimmer etal., Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y, 1989, pp.223–230; and Richards et al., FEBS Lett., 247:113 (1989). It is a highlyspecific enzyme which cleaves peptides as well as proteins (Kent et al.,supra; and Kröusslich, et al., Proc. Natl. Acad. Sci. USA, 86:807, 1989)and its specificity is determined by the interactions of the threedimensionally folded enzyme molecule forming a complex with sixconsecutive amino acid residues in the substrate polypeptide chain.Miller et al., Science, 246;1149 (1989); and Kent et al., supra.

As with all enzymes, HIV PR owes its specificity and catalytic activityto the precise three dimensional structure formed by specific folding ofthe polypeptide chain, and to precise geometric interactions in thespecific complexes formed with substrates. Fersht, in “Enzyme Structureand Mechanism”, W.H. Freeman and Company, San Francisco, 1977, pp.75–81. The observed reciprocal chiral specificities therefore show thatthe folded forms of the D- and L-enzyme molecules are mirror images ofone another in all elements of the three dimensional structureresponsible for the enzymatic activity. The extensive nature of theseinteractions implies that the two enzyme molecules are mirror images inevery respect (21), consistent with the observed equal and opposite CDspectra. Most notably, the folded form of the polypeptide backbone (i.e.ignoring the side chains) is itself a chiral entity that must exist inmirror image form in the two protein enantiomers as shown in FIG. 3.

The ribbon representation of L- and D-[Aba67,95,167,195]HIV-1 Proteasesshown in FIG. 3 is based on the X-ray crystallographic coordinates ofthe chemically synthesized enzyme when complexed with asubstrate-derived peptide inhibitor (inhibitor is not shown) asdescribed by Miller et al., Science, 246:1149 (1989). This model wasgenerated by performing a mirror image transformation of the L-enzymedata.

The folded three-dimensional ribbon “backbone” structures arenon-superimposable mirror images and contain numerous chiral elements.These are found in secondary and supersecondary structure, in thetertiary structure and in the quaternary structure as illustrated inFIG. 3. Note, for example, the relatedness of the flaps to one another;the relatedness of the helix segments to the neighboring b-strands;the-characteristic twist (right-handed, in the L-protease) of theantiparallel β-strands in each flap described by Richardson et al., in“Protein Folding”, Gierasch et al., Eds., American Association of theAdvancement of Science, Washington, D.C., 1990, pp. 5–17.; and, thehandedness of the helical segments. Since the only chiral elementintroduced in the chemically synthesized polypeptide chains is thestereochemistry at the amino acid Cα atoms (and the Cβ atoms of Thr,Ile), the data presented herein demand that all stereochemical aspectsof the folded enzyme molecule, from secondary to quaternary structure,are determined simply by the stereochemistry of the polypeptidebackbone. Thus, the present reciprocal chiral properties of thechemically synthesized enzyme enantiomers is a fundamental demonstrationthat the final folded/three dimensional structure and consequentbiological activities of this 21500 dalton homodimeric enzyme moleculeare completely determined by the amino acid sequence.

The L- and D-enzymes in this study have never seen biosyntheticconditions, and have thus never been in contact with biochemical factorsof any sort. Interestingly, the simple homodimeric enzyme moleculestudied here is formed rapidly (both folding and assembly) andaccurately even at the relatively low concentrations used in the assayconditions, as well as in more normal dialysis-from-denaturant foldingconditions. The results described herein are conclusive evidence thatwhatever their proposed role, biosynthetic factors are not required forthe formation of the correct, functional folded and assembled form ofthe protein.

The observed reciprocal chiral properties of the mirror-image enzymemolecules described herein reinforces and generalizes the chiral natureof biochemical interactions of proteins. The chiral properties of theprotein molecules themselves, which give rise to this behavior, aregiven only cursory attention in biochemical texts. We can now state,based on experimental evidence, that protein enantiomers will displayreciprocal chiral specificity in their biochemical interactionsincluding catalysis.

The observation that both enantiomers of HIV PR were equally affected bythe achiral inhibitor Evans Blue provides a number of significantimplications. First, the unnatural enantiomer of an enzyme that operateson an achiral substrate and yields an achiral product will be fullyfunctional in vivo. This aspect provides important potential therapeuticapplications. Example are carbonic anhydrase and superoxide dismutase.D-Enzymes are expected to be long lived in vivo (in an L-proteinbiosphere), since they will be resistant to naturally occurringproteases which will in general attack only proteins made up of L-aminoacids. D-proteins are comparatively less immunogenic than L-proteinsbecause long polypeptides made up entirely of D-amino acids are notprocessed and presented as efficiently by the immune system as arepolypeptides made up of L-amino acids.

D-Protein molecules have other potential practical applications.For(example enzyme enantiomers have utility as chiral catalysts in theselective production of a pure enantiomer of a fine chemical.Enantiomerically pure chemical synthesis has applications to theproduction of human pharmaceuticals. In addition, protein enantiomerscan contribute to the acquisition of phase data in X-ray crystallographyas described by Mackay, Nature, 342:133 (1989). Centro-symmetriccrystals formed by the co-crystallization of a D-, L-protein pair wouldhave greatly simplified phases, and provide more reliable X-raystructural data. At the present time D-enzymes, and D-proteins ingeneral, are accessible only by total chemical synthesis. Duringribosomal synthesis of polypeptide chains, even in vitro translationsystems, D-amino acids will not be incorporated into growingpolypeptides. Ellman et al., Science, 255:197 (1992).

4. Discussion of Examples 1–3

D- and L-forms of the enzyme HIV-1 protease are prepared herein by totalchemical synthesis. The two proteins have identical covalent structures.However, the folded protein/enzyme enantiomers show reciprocal chiralspecificity on peptide substrates. That is, each enzyme enantiomer cutsonly the corresponding substrate enantiomer. Reciprocal chiralspecificity was also evident in the effect of the enantiomericinhibitors of the HIV-1 protease enzymes prepared herein. These datashow that the folded forms of the chemically synthesized D- and L-enzymemolecules are mirror images of one another in all elements of the threedimensional structure. Enantiomeric proteins display reciprocal chiralspecificity in all aspects of their biochemical interactions, retainenzymatic activity, and provide a wide range of useful compositions asdescribed herein.

5. Synthesis and Ligation of D- and L-[Aba^(67,95) (CO—S)⁵¹⁻⁵²]₂HIV-1Protease Analogs.

FIG. 4 illustrates a schematic representation of the strategy employedfor the total synthesis of the D- and L-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂ HIV-1protease analogues. Protected D- and L-amino acids may be obtained fromthe Peptide Institute (Osaka, Japan), Peptides International(Louisville, Ky.), Bachem Bioscience (Philadelphia, Pa.) and BachemCalifornia (Torrance, Calif.) and had <0.03% of the opposite enantiomer.HPLC purified, functionalized, unprotected peptide segments, assembledby stepwise solid-phase synthesis, is reacted in the presence of 6MGuHCl to form the ligated 99-residue D- and L-[(NHCH₂COSCH₂ CO)⁵¹⁻⁵²]HIV-1 PR products. (Schnölzer et al., (1992) Science 256, 221–225.) Theboxed area of FIG. 4 represents the structure of the thioester analogueof the peptide bond Gly⁵¹–Gly⁵² at the site where the ligation occurred.The thioester serves as a link between the two D-peptides, i.e. the siteof ligation. A selenol ester linkage may also be employed for ligatingtwo D-peptides.

Two large peptide segments, i.e. [αCOSH]HIV-1 PR(1–51) and[N^(α)-BrCH₂CO]HIV-1 PR(53–99), are assembled, as illustrated in FIG. 5,in separate syntheses by a highly optimized machine-assisted SPPSprotocol using Boc-chemistry performed on a modified ABI 430Asynthesizer. (Kent et al., “Innovation & Perspectives in Solid-PhaseSynthesis”, (1992) Ed. Epton, R. SPPC Ltd. Birmingham, U.K.). Theprotocol comprised removal of the Na-Boc group with undiluted TFA (2minute total) followed by a DMF flow wash to give the TFA-peptide-resinsalt, and a single 10 minute coupling step using HBTU activatedBoc-amino acids and in situ neutralization with DIFA in DMF.Deprotection and coupling reactions are separated by a flow wash step.After purification, the monomers are separately folded by dialysis inthe presence of D- & L- MVT-101 inhibitor, respectively, to yield thehomodimeric enzymes. The milligram yields of each product are providedin FIG. 4.

The [αCOSH]HIV-1 PR(1–51) peptide may be assembled on4-[a(Boc-Gly-S)benzylI]phenoxyacetamidomethyl-resin.

The [N^(α-BrCH) ₂CO]HIV-1 PR[53–99] peptide may be prepared bybromo-acetylation of [Aba^(67,95)]HIV-1 PR(53–99)-OCH₂ Pampeptide-resin. (Yamashiro et al., (1988) Int. J. Peptide Protein Res.31, 322–334.) All peptides are cleaved and deprotected by high HFtreatment.

After preparative reverse phase HPLC purification (Vydac218TP101550-5×25 cm, 0.1% TFA/CH₃CN & 30–50 ml/min) the functionalizedpeptide segments are reacted in the presence of 6M GuHCI (in 0.1Mphosphate buffer, pH 5.3) for 48 hours to form the ligated D-andL-[(NHCH²COSCH₂CO)⁵¹⁻⁵²]HIV-1 PR monomers. FIG. 6 illustrates acomposite chromatogram showing the two purified functionalizedunprotected segments and the final (48 hour) ligation reaction product(bold) run on a Vydac 218TP5415 column eluted with 0.1% TFA (buffer A)and 0.9% TFA/CH₃CN, 1:9 (buffer B), at a flow rate of 1 ml/min.

After purification by reverse phase HPLC, the products may be separatelyfolded by dialysis in 25 millimolar phosphate buffer, pH 5.5, in thepresence of a 10-fold excess of either D- or L-[MVT-101] inhibitor(Ac-Thr-Ile-Nle-Ψ-[CH₂NH]-Nle-Gln-Arg.NH₂) (SEQ ID NO 1) to yield thehomodimeric enzymes. (Miller et al., (1989) Science 246, 1149.) Stepreaction yields for the synthesis of the D- and L-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 protease analogs are provided by FIG. 7.

Total yield with respect to the synthesis of theD-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂ HIV-1 protease analog was 48 milligrams or3.0%.

Total yield with respect to the synthesis of theL-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂ HIV-1 protease analog was 47 milligrams or2.5%.

6. Physical Characterization of the Ligation Products, D- andL-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂HIV-1 Protease Analogs

Ion spray mass spectrometry of the HPLC purified ligated products isillustrated in FIG. 8. Ion spray mass spectrometry reveals singlemolecular species in each case with observed molecular masses of10768.9±1.4 daltons (D-enantiomer) and 10769.4±0.9 daltons(L-enantiomer) [Calculated: 10763.9 daltons (monoisotopic) and 10770.8daltons (average). Minor amounts of dehydration byproducts were alsodetected. The sequences of the monomers were also examined by a newprotein ladder sequencing technique utilizing a one step laserdesorption mass spectrometric readout. FIG. 8 (A & C) illustratelabelled peaks representing a single molecular species differing in thenumber of excess protons. The observed molecular masses of the ligatedproducts is 10768.9±1.4 daltons (D-enantiomer) and 10769.4±0.9 daltons(L-enantiomer) (Calculated: 10763.9 daltons (monoisotopic) and 10770.8daltons (average)]. FIG. 8 (B & D) illustrate reconstructed mass spectrain which the raw data shown in A & C has been reduced to a single chargestate. All data points in A & C are included in the calculations and nomathematical filtering is performed. The mass regions from 10 to 11 kDare shown for clarity.

FIG. 9 illustrates the reverse phase HPLC measurements of D- &L-[Aba^(67,95), (CO—S)⁵¹⁻⁵²] HIV-1 PR ligation products. The ligatedproducts from 6M GuHCl reveal a single peak in each case. Panels A and Cillustrate the purified ligated monomers in 6M GuHCl. Panels B and Dillustrate the homodimeric enzymes folded in the presence of D- orL-[MVT-101] inhibitor, respectively, in 25 millimolar sodium phosphatebuffer, pH 5.5. Note that, after folding, a number of minor autolysisproducts are seen in both the D- and L-[Aba^(67,95) (CO—S)⁵¹⁻⁵²]₂HIV-1protease preparations. At approximately 27 minutes, minor proteolyticproducts of the MVT-101 inhibitor peptide are also seen. It would seemthat even in the presence of a large excess of inhibitor, the enzyme isstill subject to a minor degree of autolysis. The samples were run on aVydac 218TP5415 column eluted with 0.1% TFA (buffer A) and 0.9%TFA/CH₃CN, 1:9 (buffer B), at flow rate of 1 ml/min.

FIG. 10 illustrates the far-ultraviolet circular dichroism spectra ofthe folded D- and L-protease preparations. The spectra were recorded in25 millimolar sodium phosphate buffer, pH 5.5 (0.4 mg/ml protease in thepresence of inhibitor) at 25° C. in a quartz cell with a pathlength of 1millimeter. Each preparation is of equal magnitude, but opposite sign,as expected for mirror image proteins.

(Corigliano-Murphy et al., (1985) Int. J. Peptide Protein Res. 25, 225;and Zawadzke et al., (1992) J. Am. Chem. Soc. 114, 4002).

7. Characterization of the Enzymatic Activity of Ligation Products, D-and L-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂ HIV-1 Protease Analogs

FIG. 11 illustrates the enzymatic activity of the D- &L-[Aba^(67,95)(CO—S)⁵¹⁻⁵²]₂ HIV-1 PR enantiomers may be determined bytheir action on D- and L-isomers of the hexapeptide substrateAc-Thr-Ile-Nle-Nle-Gln-Arg.amide (an analog of the p24/p15 GAG viralprocessing site). The D-enzyme cleaves only the D-substrate and isinactive on the L-substrate, while the L-enzyme shows full activitytowards the L-substrate, but is inactive towards the D-substrate. Thisreciprocal chiral specificity is also evident in the effect of chiralinhibitors. As shown in the Table, D-and L-[MVT-101] inhibits thecleavage of chiral fluorogenic substrates by the D- and L-HIV-1 PRanalogues respectively, but has no effect on the action of the oppositeenantiomer. Interestingly, the achiral inhibitor, Evans Blue, whichshows mixed inhibition kinetics, inhibits both enantiomers of theenzyme.

TABLE Chiral inhibitors show reciprocal chiral specificity against D-and L-[Aba^(67.95)(CO—S)^(51–52)]₂ HIV-1 PR * L-MVT101 D-MVT101 EvansBlue D-[Aba^(67.95)(CO—S)^(51–52)]₂ + − + HIV-1 PRL-[Aba^(67.95)(CO—S)^(51–52)]₂ − + + HIV-1 PR

The D- and L-enzymes were separately assayed by the fluorogenic assaymethod using the corresponding chiral substrate, in the presence of5×1C₅₀ concentration of inhibitor. The fluorogenic assays were performedwith 15 ul aliquots (corresponding to 1.75 (±10%) mg protein) of eachenzyme enantiomer in 100 millimolar MES buffer pH 6.5 added to asolution of 50 mM D- or L-fluorogenic substrate in the MES buffer. Thesubstrate sequence was 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO₂)-Gln-Argamide (SEQ ID NO 2): it was synthesized with either D- or L-amino acidderivatives to provide the appropriate enantiomeric forms. The inhibitorEvans Blue is a non-peptide, achiral mixed competitive-uncompetitiveinhibitor of the HIV-1 PR enzyme.

The enzymatic activity of the [Aba^(67,95),(CO—S)⁵¹⁻⁵²]₂ HIV-1 PRenantiomers with respect to the D- and L-isomers of the substrateAc-Thr-Ile-Nle-Nle-Gln-Arg.amide (SEQ ID NO 3) may be measured asfollows. The substrate (1 mg/ml) is treated with enzyme (0.1 mg/ml) atpH 6.5 (MES buffer, 100 mM) for 30 minutes at 37° C. An aliquot of thereaction mixture is then chromatographed (Vydac 218TP5415 RP HPLCcolumn) with a linear gradient, 0–40%, of buffer B (0.09% TFA/CH₃CN,1:9) in buffer A (0.1% TFA) over 20 minutes (flow rate 1 ml/min,A^(214nm)). The peptide products are identified by ion spray MS as(H)-Nle-Gln-Arg.amide (m/z: 415.0—early eluting) and Ac-Thr-Ile-Nle-(OH)(m/z: 388.0—late eluting). Minor impurities present in the substratepreparations due to unpurified peptides are not cleaved. Panel Aillustrates D-substrate only; panel B illustrates L-substrate only;panel C illustrates D-substrate plus D-enzyme; panel D illustratesD-substrate plus L-enzyme; panel E illustrates L-substrate and L-enzyme;panel F illustrates L-substrate plus D-enzyme.

8. Discussion of Examples 4–7

The HIV-1 protease enzyme exists as a homodimeric structure. It is ahighly specific enzyme and this specificity and its catalytic activitydepend on a precise 3-D structure being formed between the folded dimerand six residues of the substrate molecule. The observed reciprocalspecificities, therefore, show that the folded forms of the D- andL-enzyme molecules are mirror images of each other in all elements ofthe 3-D structure responsible for their enzymatic activity. This isconsistent with their observed CD spectra.

The 3-D structure of a folded enzyme molecule contains numerous chiralelements in secondary and supersecondary structure, in tertiarystructure and in quarterny structure, as illustrated in FIG. 3. Sincethe only difference between the synthetic D- and L-polypeptide chains isthe stereochemistry of the a-carbon atoms (and the Cβ atoms of Ile andThr) of the amino acids, it is concluded that the stereochemistry of thebackbone determines all aspects of higher structure in this protein.

The observations of reciprocal chiral specificity in the enzymaticactivity of the D- and 1-HIV-1 proteases disclosed herein, serve togeneralize and emphasize the chiral nature of the biochemicalinteractions of proteins. The large amounts of high purity D- andL-enzyme enantiomorphs prepared using the chemical ligation method willallow a thorough experimental evaluation of the use of D-, L-proteins inX-ray crystallography.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications can be effectedwithout departing from the true spirit and scope of the invention.

1. A method for screening a library for identifying an active enantiomerof a chiral drug candidate having activity with respect to an L-protein,wherein the activity is selected from the group consisting of inhibitionand binding, the method comprising the following steps: Step A:providing a D-protein, the D-protein being an enantiomer of theL-protein, and a chemical library comprising chiral compounds; Step B:contacting one or more compounds from the library under suitableconditions with the D-protein of said Step A and identifying at leastone compound having activity to said D-protein; Step C: providing one ormore enantiomer compounds of said at least one compound identified inStep B, and contacting said L-protein with said one or more saidenantiomer compounds; and Step D: identifying at least one enantiomercompound provided in Step C that binds to said L-protein in order toverify the activity of said enantiomer compound that binds to saidL-protein.
 2. A method as described in claim 1, wherein the L-proteincomprises a receptor or receptor binding site.
 3. A method for screeninga library for identifying an enantiomer compound that binds to anL-protein, said method comprising: Step A: contacting one or morecompounds from a chemical library with a D-protein, wherein saidD-protein is an enantiomer of said L-protein; Step B: identifying atleast one compound that binds to said D-protein; Step C: providing oneor more enantiomer compounds of said at least one compound identified inStep B, and contacting said L-protein with said one or more enantiomercompounds; and Step D: identifying at least one enantiomer compoundprovided in Step C that binds to said L-protein.
 4. The method of claim3, wherein said L-protein comprises a receptor or a receptor bindingsite.
 5. The method of claim 4, wherein said L-protein is an L-enzyme.6. The method of claim 3, wherein said chemical library comprises chiralcompounds.
 7. The method of claim 6, wherein said chiral compoundscomprise compounds having random chirality.
 8. The method claim 3,wherein said chemical library comprises natural compounds.
 9. The methodof claim 3, wherein said chemical library comprises synthesizedcompounds.
 10. The method of claim 3, wherein said D-protein is producedby chemical ligation of D-peptide segments.
 11. The method of claim 4,further comprising the additional step of screening said enantiomercompound identified in Step D for agonist or antagonist activity againstsaid L-protein.
 12. A method for screening a library for identifying anenantiomer compound that binds to an L-protein, said method comprising:Step A: contacting one or more compounds from a chemical library withboth said L-protein and a D-protein, wherein said D-protein is anenantiomer of said L-protein; Step B: identifying at least one compoundthat binds to said L-protein and/or to said D-protein; and Step C:optionally, if said at least one compound identified in Step B binds tosaid D-protein, then providing one or more enantiomer compounds of saidat least one compound identified in Step B, and contacting saidL-protein with said one or more enantiomer compounds and identifying atleast one enantiomer compound that binds to said L-protein.